Biz teknik yardım için Tıp Keio Üniversitesi School Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi ve Rei Ohno teşekkür ederiz. Bu çalışma kısmen sağlığı araştırma sonuçlarının pratik kullanımını hızlandırmak için bir Ar-Ge Sistemleri destek programı ve Rejeneratif Tıp Gerçekleşmesi için Karayolları Programı tarafından finanse edildi.

1. aktiflediği insan T hücrelerindeki hazırlayın <ol><li> Damarından bir donörden tam kan heparinize 10 ml toplayın. <ol><li> Venipunktür önceden donörlerin aydınlatılmış onam. Venipunktür kalifiye ve eğitimli personel tarafından yapılmalıdır. </li><li> Steril ekipman ve aşağıdaki uygun biyogüvenlik kurallarına elde edilen kan örnekleri taşıyınız. </li></ol></li><li> 10 ml D-PBS ile bütün kan heparinize 10 ml seyreltik (-). </li><li> Yeni 50-ml konik tüp içine 15 ml Ficoll solüsyonu (Ficoll Paque PREMIUM) koyun. </li><li> Adım 1.3 Ficoll çözeltisi üzerine Adım 1.2 hazırlanan 20 ml kan çözümü Katman. Elektrikli bir pipet kullanılarak, kan solüsyonu tüpün iç duvarı boyunca yavaş yavaş Aşama 1,2 çalıştırmak hazırlandı sağlar. Kan çözümü ve Ficoll çözüm karıştırmak için dikkatli olun. </li><li> Oda sıcaklığında 30 dakika boyunca 400 x g&#39;de santrifüjleyin. &quot;Yavaş&quot; santrifüj yavaşlama hızını ayarlayın. Not: Santrifüj işleminden sonra, beyaz bir incetabaka bir üst sütlü plazma tabakası ve alt net bir Ficoll katmanı arasında ortaya çıkar. Bu beyaz ince tabaka mononükleer hücreleri içerir. </li><li> Bir 1 ml pipet ile yeni bir 50-ml&#39;lik konik tüpe tek çekirdekli hücre katmanı aktarın. Ficoll çözüm dahil vermemek için dikkatli olun. </li><li> D-PBS (-) ekleyin, oda sıcaklığında 5 dakika boyunca 200 x g&#39;de, toplam 10 ml&#39;lik bir hacme ve santrifüj mononükleer hücrelerine temin etmektedir. </li><li> Süpernatantı atın. Tek-çekirdekli hücreleri, ve oda sıcaklığında 5 dakika boyunca 200 x g&#39;de santrifüj (-) D-PBS, 10 ml ekleyin. </li><li> Süpernatantı atın toplanan mononükleer hücrelere KBM502 orta 1 ml ekleyin ve hemasitometre kullanarak hücreleri saymak. X 10 6 5 üzerinde tek çekirdekli hücreler, uygun bir ayırma Ficoll kullanılarak, tam kanın heparinize 10 ml elde edilebilir. </li><li> D-PBS içinde 10 ug / ml&#39;lik bir konsantrasyonda, bir anti-insan CD3 antikoru solüsyonu hazırlayın (-). Sörf emmek 6-çukurlu plaka anti-insan CD3 antikoru solüsyonu ilaveHer bir ası de ve en az 30 dakika boyunca% 5 CO2 inkübatörde 37 ° C&#39;de inkübe çanak. </li><li> Her bir anti-insan CD3 antikor çözüm çıkarın ve D-PBS ile bir kez yıkanır (-) tohum hücreleri hemen önce. </li><li> KBM502 ortam içinde anti-insan CD3 antikoru ile kaplanmış 6-yuvalı plakalar üzerinde 2.5 x 10 5 hücre / cm2 yoğunluğunda Aşama 1,9 hazırlandı Tohum mononükleer hücreleri. T hücreleri konfluansa ulaşana kadar bir ortam değişikliği olmadan 3-7 gün boyunca% 5 -CO 2 inkübatörde 37 ° C&#39;de inkübe edilir. Bu dönemde, T-hücreleri, hem süspansiyon haline getirilmiş ve yapışan hücreler bulunmaktadır. </li></ol> SeV Vektörler 2. Infect İnsan T hücrelerinin <ol><li> 3-7 gün kültürden sonra, pipetleme orta mevcut aktive edilmiş T hücreleri toplamak. Oda sıcaklığında 5 dakika boyunca 200 x g &#39;de 15 ml konik tüp ve santrifüj aktarın. </li><li> Süpernatantı ve 1 ml taze KBM502 orta ekleyin. Hücreleri saymak ve 1 plaka.5 x 10 6 hücre, bir anti-CD3 antikoru ile kaplanmış 6-çukurlu plakanın her oyuğuna (2 ml taze KBM502 ortamı içerisinde). </li><li> Buz üzerinde Zeiss OCT3 / 4-SeV / TSΔF, Sox2-SeV / TSΔF, Klf4-SeV / TSΔF ve c-myc (HNL) -SeV / TS15ΔF arasında SeV çözüm çözülme. </li><li> Zeiss OCT3 / 4-SeV / TSΔF, Sox2-SeV / TSΔF, Klf4-SeV / TSΔF ve c-myc (HNL) ihtiva eden SeV çözümler ekleyin -SeV / TS15ΔF ayrı ayrı gözlere, enfeksiyon çokluğunda her (MOI) Bir başka 24 saat süreyle% 5 -CO 2 inkübatörde 37 ° C&#39;de inkübe 10. bölgesinin. </li></ol> 3. İnsan T Hücreleri SeV Vektörler kaldır <ol><li> 24 saat enfeksiyondan sonra, bir pipetle enfekte hücreleri toplamak. Yapışan hücreler varsa, onlar kolayca defalarca yukarı ve aşağı onları pipetleme ayrılabilir. Oda sıcaklığında 5 dakika boyunca 200 x g &#39;de 15 ml konik tüp, ve santrifüj aktarın. </li><li> SeV vektörleri içeren süpernatant kaldırmak ve 2 ml taze KBM502 orta ekleyin. EAC içinde Replate hücrelerkuyuların h. Ek bir 24 saat boyunca bir% 5 -CO 2 inkübatörde 37 ° C&#39;de inkübe edilir. </li></ol> Bir Matrigel Katman 4. reseed Hücreler <ol><li> 4 ° C &#39;de (Bu çalışmada kullanılan özel markasıyla Malzeme Listesi bakınız) ve DMEM / F12 ortamı içinde 10 ml 0.2 ml eritilmesi çözülme matris jel bazal membran matrisi çözeltisi hazırlayın. Not: The Matrix jeli (bu çalışmada kullanılan belirli bir marka için malzemeler listesi bakınız) 4 ° C&#39;de tamamen çözülmüş olması O / N çözülme gerekiyor. Kullanmadan önce kadar buz üzerinde tutun. </li><li> 100 mm&#39;lik tabaklara tabak başına Levha 5 mi bazal membran matrisi çözeltisi ve tohum hücreleri önce en az 30 dakika boyunca oda sıcaklığında bekletin. En az iki adet 100 mm çanağı bir deney için ihtiyaç vardır. </li><li> , Pipetleme SeV enfekte olmuş hücreleri toplamak, oda sıcaklığında 5 dakika boyunca 200 x g&#39;de 15 ml konik tüp, ve santrifüj aktarın. </li><li> Süpernatantı ve 1 ml taze KBM502 orta ekleyin. Ce sayısınıHemasitometre kullanarak lls Adım 4.2 ve plaka hazırlanan 100 mm yemeklerinden bazal membran matris çözümü kaldırmak 1 × 10 5 ve 1 × 10 6 hücre 100 mm bazal membran matris kaplı üzerinde (10 ml taze mTeSR orta olarak) yemekler. Son olarak, koloniler kolayca çekilen hangi plakayı kullanın. </li><li> % 5 CO 2 inkübatör O / N 37 ° C &#39;de çanak inkübe edin. </li><li> 10 orta değiştirmek mi her gün mTeSR orta eti. Not: enfeksiyondan sonra yaklaşık 20-30 gün, yakından embriyonik kök hücreler (EKH) benzer koloniler görünecektir. </li></ol> 5. T-hücresinden alınan iPSCs aç <ol><li> 4 ° C &#39;de (Bu çalışmada kullanılan özel markasıyla Malzeme Listesi bakınız) ve DMEM / F12 ortamı içinde 10 ml 0.2 ml eritilmesi çözülme matris jel bazal membran matrisi çözeltisi hazırlayın. </li><li> Levha 1, 6-çukurlu plaka içinde çukur başına bazal membran matrisi çözeltisi ve en oda sıcaklığında bırakınkoloniler almadan önce en az 30 dakika. </li><li> MTeSR ortamının 20 ul, 96 oyuklu bir plaka Dolgu ile sabitleştirilir. </li><li> 20 ul pipet kullanarak stereomicroscope altında EKH benzer koloniler seçin. Not: Şekil 1B gösterildiği gibi EKH ve iPSCs kolonileri yuvarlak ve düzdür. </li><li> Aşama 5.3 mTeSR ortamı ile doldurulmuş 96 oyuklu bir plakaya transfer seçilmiş kolonileri. </li><li> 200 ul pipet kullanarak stereomicroscope altında küçük öbekler halinde koloni kırmak için aşağı yukarı pipet ve dikkatle her kuyuya mTeSR orta 200 ul ekleyin ve. 5.2 de hazırlanan gelen bazal membran matrisi çözeltisi çıkarın ve mTeSR orta 2 ml bazal membran matris ile kaplanmış 6-çukurlu plakanın bir oyuğuna, her bir koloni koyun ile sabitleştirilir. </li><li> 2 orta değiştirmek mi her gün mTeSR orta eti. </li></ol> 6. T hücre-türevi iPSCs korumak T hücresi kaynaklı iPSCs korunabilirve insan ve insan iPSCs EKH aynı teknikler kullanılarak muhafaza edilmiştir. IPSC kolonileri büyümek kez aynı prosedürü tekrar ederek büyük yemeklerin içine genişletin. <ol><li> Her 1-2 gün mTeSR orta değiştirin. </li><li> Koloniler Konfluent hale geldiğinde her 5-7 gün, insan iPSCs ve üreticinin talimatlarına göre insan EKH için ayrılma çözümü kullanarak geçiş hücreleri. </li></ol>

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Yazarlar hiçbir rakip ya da ifşa etmek çıkarları çatışan var.

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPSCs rejeneratif tıp 1-3 için hücre çığır kaynağı olarak büyük ilgi çekmiştir. Bugüne kadar, iPSCs üretilmesi için çeşitli yöntemler 4,5 bildirilmiştir. Bunlar arasında, insan T hücrelerinden üretilen iPSCs çünkü cep örnekleme 6-8, daha az invazif yöntemin özel ilgi olmuştur. Buna ek olarak, T hücrelerinden türetilen iPSCs T hücre reseptörü (TCR) geninin yeniden düzenlenmesi içerir ve dolayısıyla antijen-spesifik T hücrelerinin 9,10 kaynağıdır. Bu nedenle, T-hücresi türevli iPSCs üreten güvenli rejeneratif tıp devam edebilmek için yararlıdır. Bu yöntem, öngörülemeyen patojenite riskini azaltma kavramına dayanmaktadır. IPSCs güvenli klinik uygulama için bu patojenlere 11 maruz kalma riskini azaltmak için çok önemlidir. Daha önce pluripotent kök hücrelerin bir çok kültür sistemlerinde, fetal sığır serumu ve besleme hücreleri esas reaktif olarak kullanılmaktadır12 s. IPSC üretimi öngörülemeyen patojenik riskini azaltmak için Ancak, kültür sistemi, bu reaktiflerin iki çıkarılması tercih edilir. Buna ek olarak, bu yöntem, hasta ve besleyici hücreler zahmetli preparatından invaziv hücre örnekleme önüne geçme avantajına sahiptir. T hücresi türevli iPSCs önce hastalığı araştırma 13,14 başarıyla kullanılmıştır, bu yöntem, aynı zamanda hastaların hastalığa özgü iPSCs üretilmesi için kullanılabilir ve yararlıdır. Bir gen taşıyıcı olarak Sendai virüsü (SeV) vektörü kullanılarak T hücre yeniden programlama yöntemleri arasında, yüksek verimlilik, 7,16 ile iPSCs üreten bir yöntemdir. SeV tek iplikli bir RNA virüsüdür ve replikasyon için bir DNA faz gerek yoktur, çünkü ek olarak, iPSC üretimindeki kullanımı konakçı genomu 17-19 kırılma önler. Bu nedenle, serum-fr insan periferik T hücrelerinden iPSCs üretilmesi için protokoller kurdukee ve matrigel, mTeSR ortamı bir kombinasyonu kullanılarak besleyici içermeyen koşulları ve SeV vektörler.

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

Son zamanlarda, iPSCs rejeneratif tedaviler için hücrelerin yeni kaynağı olarak dikkat çekmiştir. Üreten iPSCs için ilk yöntem invaziv biyopsi ve transgenlerin retroviral genomik yerleştirilmesi ile elde edilen dermal fibroblastlar dayanıyordu olsa da, bu dezavantajları önlemek için birçok çaba olmuştur. İnsan periferal T-hücreleri iPSCs üretilmesi için benzersiz bir hücre kaynağıdır. T hücrelerinden türetilen iPSCs T hücre reseptörü (TCR) genlerinin yeniden düzenlemeleri içerir ve antijen-spesifik T hücrelerinin bir kaynağıdır. Buna ek olarak, genom içinde T hücresi reseptörü yeniden düzenleme Bireysel hücre serilerinin etiket ve nakledilen ve donör hücreleri ayırt etmek için bir potansiyele sahiptir. IPSCs güvenli klinik uygulama için, zararlı ajanlara yeni oluşturulan iPSCs ortaya çıkma riskini en aza indirmek için önemlidir. Fetal büyükbaş hayvan serumu ve besleme hücreleri pluripotent kök hücre kültürü için gerekli olmasına rağmen, bu riskini azaltmak için kültür sistemi kaldırmak için tercih ediliröngörülemeyen patojenli. Bu gidermek için, tanımsız patojenlere iPSCs ortaya çıkma riskini azaltmak için Sendai virüsü kullanılarak insan periferik T hücrelerinden iPSCs üretilmesi için bir protokol kurduk. Sendai virüsü taşıma, uygun biyogüvenlik düzeyi ile ekipman gerektirir rağmen, Sendai virüsü bozar genom ekleme olmadan T hücrelerinin aktive henüz yüksek verimlilik ile. Bu protokol, aktive edilmiş T hücre kültürü ve Sendai virüsü bir kombinasyonu kullanılarak besleyici içermeyen koşullar, insan periferik T hücrelerinden iPSCs üretilmesini göstermektedir.

<html> Bu protokol kullanılarak, kullanıcı kararlı bir şekilde insan periferik T hücrelerinden iPSCs elde edebiliyoruz. . Birkaç geçiş sonrası tespit olmayan birden çok verici olgu ve sev arasında bir hücrenin yeniden programlanması verimliliği% 0.005 16 Şekil 1A T üretilmesi için prosedürün şematik hücre türevli gösterir - SeV ve Matrigel ile T hücrelerinden iPSC üretimi yaklaşık% 0.002 gösterdi Matrigel ve mTeSR ortamı kullanılarak besleyici içermeyen koşullarda iPSCs. Çevresi 20-30 gün sev ile enfeksiyondan sonra, iPSC koloniler ESC koloni gibi morfolojisi (Şekil 1B) tarafından kabul edilmektedir. Immünofloresan boyama tipik pluripotent hücre belirteçleri ekspresyonunu gösterdi (Nanog, Zeiss OCT3 / 4, SSEA4, TRA-1-60 ve TRA-1-81), T hücresi kaynaklı iPSCs besleyici içermeyen koşullar (Şekil 1C) altında elde edilen. &quot;Width =&quot; 700 &quot;/&gt; Şekil 1. (A): Bu çalışmada, besleyici içermeyen koşullar altında yeniden programlama T hücreleri için protokol şematik. (B): besleyici içermeyen koşullar altında kan örneği gün sonra 27 bulunan tipik bir ESC benzeri iPSC koloni. (C)., T hücresi kaynaklı iPSCs ALP boyanması ve pluripotense ve yüzey markörleri için immünofloresan boyama (Nanog, Zeiss OCT3 / 4, SSEA4, TRA-1-60 ve TRA-1-81) besleyici içermeyen koşullar altında elde <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">edin Bu rakamın büyük halini görmek için buraya tıklayın.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Besleyici içermeyen şartlar Sendai virüs kullanılarak insan periferik T hücrelerinden uyarılmış pluripotent kök hücrelerin üretilmesi

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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JoVE Journal

Developmental Biology

11.9K Görüntüleme.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Video: Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Bütünleşmeyen episomal plasmidler kullanılarak dondurulmuş Buffy tüylerdeki uyarılmış pluripotent kök hücrelerin üretilmesi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Gelişim Biyolojisi

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Saç türetilmiş keratinositlerden Kullanarak Entegrasyon serbest İnsan uyarılmış pluripotent kök hücrelerin üretilmesi

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Insan pluripotent kök hücreleri gelen melanosit besleyici içermeyen türetilmesi

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

İnsan Melanom tümör-süzücü lemfositler ile ilgili uyarılmış pluripotent kök hücrelerin üretilmesi

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Sendai Virüs ve santrifüj kullanılarak Kan Hücreleri indüklenmiş pluripotent kök hücre Üretimi

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Epizomal vektörlerin kullanılması insan periferik kan mononükleer hücrelerinden entegrasyon içermeyen uyarılmış pluripotent kök hücrelerin üretilmesi

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Tanımlı ve hepatosit benzeri Hücrelerinin Ölçeklenebilir Nesil insan pluripotent kök hücreleri gelen

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

Periferal Kan Türevi İnsan Kaynaklı Pluripotent Kök Hücrelerden Kondrositlerin Farklılaşması

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Standart ve tekrarlanabilir ön türü beyin Organoids insan nesil Pluripotent kök hücreler indüklenen

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

3B cilt Organoid kablosu kan kaynaklı nesil Pluripotent kök hücreler indüklenen

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...